The role of the Met tyrosine kinase receptor in skin maintenance and regenerationJolanta Chmielowiec1Zusammenfassung2Introduction Wound healing in the skin 23534#4274551#2335Mammalian skin6534#328The tyrosine kinase receptor Met7Met signal transduction8534#372534#6089550#226Met signalling during development10534#20111Met function in the adult The aim of the study12ResultsExpression of Met and HGF/SF in the skin and during skin wound healing12538#18913451#190Generation of mice deficient in Met in keratinocytes 14476#38215393#294Met signaling during generation and maintenance of the skin16382#658507#43417Wound closure in conditional Met mutant mice18380#672390#14819534#69620584#554Contribution of cells in the hyperproliferative epithelium21436#419481#30022Scratch-wound healing of Met mutant keratinocytes in cell culture585#21323587#47424612#376Cytoskeleton rearrangement in cultured scratchwounded keratinocytes 476#94125Signal transduction in primary keratinocytes26586#731Discussion26Conditional mutagenesis to investigate Met function in the skin27The role of the tyrosine kinase receptor Met in the skin28Only non-recombined cells contribute to wound healing29The role of HGF/SF and Met in development and regeneration30Only Met-positive keratinocytes contribute to healing of scratch-wounds in vitro31The Met receptor as a therapeutically targetMaterials and Methods32Extraction and purification of DNA3334Polymerase chain reaction (PCR)Southern blotting35Cell culture36Wounding of skinImmunhistochemical techniques37383940414243Protein biochemistry 4445534#216464748AbbreviationsReferencesErklärungPublicationsenTable of contentsHelp
Results
Expression of Met and HGF/SF in the skin and during skin wound healing

The role of Met and HGF/SF has been recently implicated in the skin and in the hair follicle cycle, as shown by overexpression experiments and skin organ culture (Lindner et al., 2000). Theses experiments however, do not show the endogenous in vivo function of Met signaling system in the skin. In order to determine the precise expression profile of Met and HGF/SF in the skin, immunohistological analysis on tail skin paraffin sections was performed. The immunohistological analysis demonstrated that Met is expressed in the epidermis and its ligand, HGF/SF, in the dermis, which may suggest a possible role of this signaling system in the skin (Fig.8A, B). This is also in an agreement with the literature (Lindner et al., 2000). The two expression domains of HGF/SF and Met are close to each other and could function in a paracrine manner.

Figure 8. Expression of the Met tyrosine kinase receptor and its ligand, HGF/SF, in the skin. A. Immunofluorescence staining using Met antibody on a tail skin section. Met is detectable in the epidermis. B. Immunofluorescence staining using HGF/SF antibody on a tail skin section, showing HGF/SF protein expression in the dermis, associated with loosely packed fibroblasts. C. Hematoxylin/eosin staining on a tail skin section. This staining helps to recognise the morphology of tail skin. The round structure in the dermis is a hair follicle.

Furthermore, immunofluorescent staining of phosphorylated Met on skin sections was performed to investigate the functional state of the expressed Met receptors. The data from these experiments revealed that activated Met was present in both, epidermis and hair follicles, including hair bulge stem cells (Fig.9A, B). In addition, phosphorylated Met was continuously detected in whole epidermis at both early (P5, Fig.9B) and later stages (P32, Fig.9A) of skin. Using antibodies against CD34, which stain hair bulge stem cells and hematopoietic cells, colocalization with phosphorylated Met in the hair bulge stem cells could be observed (Fig.9A, B). These results clearly show that activated Met is present in the epidermis and hair follicles, further implicating a potential functional role of Met/HGF/SF signaling system in the skin.

Figure 9. Expression of activated Met in the skin. A, B Immunostaining of skin section of the age P32 (A) and P5 (B) with anti phosphoMet (red) and anti CD34 (green) antibodies. CD34 is a marker of hair bulge stem and hematopoietic cells. The merged fluorescence shows that Met and CD34 are colocalized in the bulge stem cells. Scale bar 20μm

Generation of mice deficient in Met in keratinocytes

The previous expression studies point to a potential role of HGF/SF and the Met receptor in the skin. To further analyse the importance of the Met signaling system in the skin and in physiological processes such as skin wound repair, conditional mutagenesis of Met in the keratinocytes of mice was employed. The keratin 14-cre (K14-cre) mice were used, which express the cre recombinase in the epidermis starting on embryonic day 15, to introduce a null mutation into the Met locus in the skin. Keratin 14, and thus K14-cre, are known to be ubiquitously expressed in hair follicles and basal cells of the epidermis, as well as in tongue and esophagus(Huelsken et al., 2001). First, K14cre mice were crossed with the conventional Met null mutation, Metnull (Bladt et al., 1995), to generate animals with a K14-cre; Metnull/+ genotype. Then, these animals were mated with “floxed” Met mice, Metflox/flox (Borowiak et al., 2004), to obtain animals with the critical K14-cre; Metnull/flox genotype, in which one allele of Met corresponds to the conventional null mutation, the other to a ‘floxed’ allele. Following K14-cre-mediated recombination, the exon encoding the essential ATPbinding site of Met was removed in the Metflox allele of the skin, and a nonfunctional null allele, which was denoted as MetΔ, was generated (see structures of non-recombined and recombined alleles in Fig.10A). This breading procedure ensured that only a single allele needs to be recombined by cre to obtain a complete ablation of Met function in the epidermis. The generated K14-cre; Metflox/null mice are subsequently termed “conditional Met mutant mice”.

In these experiments, control animals were heterozygous Metflox/+ mice that also carried the K14cre allele or heterozygous Metflox/+ and Metnull/+ mice without cre. Such controls are essential, e.g. in order to check effects caused by cre protein by itself. These control mice did not show any overt abnormalities when compared to wildtype mice. This also indicated that the inserted loxP site in the Met locus does not interfere with Met function in vivo. However, efficient recombination of Met was observed in the epidermis of the K14cre; Metflox/null mice. Southern blot analysis demonstrated that virtually all (95%) of the cells in the epidermis had recombined the Metflox allele already at embryonic day 17.5 (MetΔ, Fig.10B). A similar high level of cells containing the recombined allele was observed in the epidermis of young and adult animals, e.g. at P8 and at 12 weeks (Fig.10B). In other epithelial tissues like pancreas, lung and liver, recombination of Met was not observed (Fig.10C).

Figure 10. Generation of skinspecific Met mutant mice. A. Schematic representation of nonrecombined and recombined alleles of Met. Exon 15 of the Met gene that encodes the ATP-binding site (red box) was flanked by loxP sites (triangles) and is excised after K14cre-induced recombination. Blue boxes indicate exons 14 and 16. The sizes of the restriction fragments generated by BamHI digest before and after recombination are indicated. B, BamHI; P, Pst. B. Southern blot analysis of epidermis from control and conditional Met mutant mice of different ages (12 weeks old, E17.5, P8). C. Southern blot analysis of different organs of conditional Met mutant mice.

The recombination introduced by the K14cre allele that occurred in virtually all epidermal cells, was also shown histologically using the Z/AP reporter mice. In such mice, activation of alkaline phosphatase is detectable by yellow NBT/BCIP staining in recombined cells, whereas blue LacZ staining is observed in the nonrecombined cells (Lobe et al., 1999). K14-cre mediated expression of alkaline phosphatase demonstrated that the vast majority of the cells in the epidermis and hair follicles had undergone recombination, and only very small groups of nonrecombined cells were detectable (Fig.11A, the enlarged picture shows a group of nonrecombined cells, Fig. 11B-C). The blue LacZ staining was observed in cells that do not express K14cre, i.e. in the dermis and the arrector pili muscles, which anchor in the dermis and insert onto the sheath of hair follicles. The nonrecombined cells in the epidermis were observable with a frequency of approximately 5%, which is comparable with the K14-cre mediated recombination in the Met locus, as detected by Southern blotting.

The high efficiency of recombination allowed further examination of Met deficient keratinocytes in the conditional Met mutant mice.

Met signaling during generation and maintenance of the skin

The conditional Met mutant mice were born in numbers predicted by the Mendelian ratio. These mice were fertile, had a normal life span and showed no overt abnormalities in the skin and other epithelial organs. The appearance of skin and hair in Met mutant mice was examined more closely by histology at birth and afterwards. No gross morphological changes in the epidermis could be detected when compared with control mice. The thickness of the epidermis was comparable and did not display any pathological alterations in the mutants. There were no apparent changes in hair cycle progression, when control and conditional mutant mice were compared. For instance, the first and second anagen phases occurred at P5 and P30, respectively. Catagen and telogen occurred at P18 and P20 (Fig.12, Paus and Cotsarelis, 1999). The conditional Met mutant mice were kept for nearly 2 years and unusual hair loss or other changes in the appearance of the aged skin could not be observed.

Further immunohistological analysis of the conditional Met mutant skin did not reveal essential changes in expression of markers for terminal differentiation in the epidermis, when compared to controls. Keratin 10 and loricrin continued to be expressed in the upper, differentiated layer of the mutant and control epidermis (Fuchs et al., 1992; Byrne et al., 1994). Keratin 6 was detectable only in the hair follicles in the mutants and controls (Fig.13, Fuchs, 1990;Wankell et al., 2001).

Figure 13. Immunohistological analysis of the skin in conditional Met mice A. Immunohistological staining for keratin 10 and keratin 6 on dorsal skin paraffin sections of 2 months old mutant and control mice. Keratin 6 is constitutively expressed in the outer root sheath of hair follicles and is observed in both, control and mutant mice. Keratin 10 is present in the differentiated, upper layers of the epidermis. B. Immunohistological staining for loricrin on dorsal skin sections of control and mutant mice. Loricrin is expressed in upper layers of the epidermis. There are no differences in expression of these proteins in the skin between mutant and control.

The data from these experiments clearly indicate that Met is not essential for the development and the maintenance of both, the epidermis and the hair.

Wound closure in conditional Met mutant mice

In previous studies, Met signaling has been shown to be important during liver regeneration, and it was found that the expression levels of Met and HGF/SF increased after injury of many organs (Ohmichi et al., 1996; Kawaida et al., 1994; Nakamura et al., 2000; Borowiak et al., 2004). Wound healing of the skin is an important regenerative process in mammals (Martin, 1997; Werner and Grose, 2003), but our knowledge in this area is still rudimentary.Therefore, the function of Met in the skin under stress conditions was investigated, specifically the effect of the absence of Met during skin would healing.

Fullthickness dorsal skin wounding in control and conditional Met mutant mice was performed in a way that epidermis and underlying dermis are destroyed (Werner et al., 1994). For these experiments, only males at an age of 12 weeks were used to exclude differences caused by gender variation. First HGF/SF and Met expression during the wound healing process was analyzed, i.e. 1 to 10 days after the injury, by in situ hybridization on frozen skin sections. HGF/SF expression was initially detected in the dermis adjacent to the wound clot, an area where inflammatory cells accumulate and infiltrate the lesion (Fig.14B, only the left halves of the wounds are shown; see scheme of entire wound in Fig.14A). Three days after injury, HGF/SF was strongly upregulated in the hyperproliferative epithelium (HE) at the edges of wounds (Fig.14C). At this time point, the development of newly formed epithelium was visible. HGF/SF was also expressed in hair follicles of skin wound sections but not in the epidermis (Fig 14B, C) (Lindner et al., 2000).The receptor tyrosine kinase Met was also shown to be strongly expressed in the hyperproliferative epithelium during the wound repair process (Fig.14E, F, (Cowin et al., 2001). Of note, Met was present in the unwounded epidermis and hair follicles shown by in situ hybridization (Fig.14E, F), further confirming the immunohistological data. Collectively, the data indicate that during wound healing, HGF/SF and Met may signal in an autocrine manner in the hyperproliferative epithelium, and that Met signaling is upregulated during the repair process, suggesting an important role during skin repair.

Figure 14. Expression of HGF/SF and Met during wound healing. A. Scheme of an entire wound 3 days after injury. Keratinocytes (red) at the wound edge proliferate and migrate down the injured dermis to form the socalled hyperproliferative epithelium (HE, marked by arrow). G, granulation tissue; D, dermis; F, fatty tissue; Es, eschar. B and C. In situ hybridisation of wounded skin with HGF/SF probe 1 day (B) and 3 days (C) after injury. HGF/SF is expressed in the dermis close to the clot at day 1. At day 3 after wounding, HGF/SF is highly expressed in the hyperproliferative epithelium (HE). E and F. In situ hybridization with the Met probe 1 day (E) and 3 days (F) after injury. Met is expressed in the epidermis and in the hyperproliferative epithelium (HE) at day 3 following wounding. D and G In situ hybridization with sense probes of HGF/SF (D) and Met (G). Scale bar, 50μm

The main structure responsible for wound closure is thought to be the hyperproliferative epithelium (Martin, 1997; Singer and Clark, 1999; Santoro and Gaudino, 2005). First, K14-cre was characterised in the skin wounds using Z/AP reporter mice, in order to determine whether K14cre is expressed in the hyperproliferative epithelium and could be used for wound healing experiments. This experiment demonstrated successful recombination introduced by K14-cre, shown by activation of alkaline phosphatase and detected by yellow NBT/BCIP staining, in virtually all epidermal cells as well as in the wound epithelium (Fig.15A). Immunohistological analyses using keratin 14 antibodies confirmed that keratin 14 was strongly expressed in the hyperproliferative epithelium (Fig.15B).

Figure 15. Expression of K14cre during wound healing. A. Double staining of alkaline phosphatase and βgalactosidase activity of wound section from Z/AP; K14cre mice. K14cre-induced recombination is observed in the unwounded epidermis and in the hyperproliferative epithelium. B. Immunohistological analysis of a wound section from control mice using antibodies directed against keratin 14 (red) and fibronectin (green). Scale bar, 100μm

The histology of wounds 315 days after the injury of control and conditional Met mutant mice was analysed. The hyperproliferative epithelium was thinner and its formation was delayed in the conditional Met mutant mice, assessed by hematoxilin/eosin (Fig.16A, D) and by Masson trichrome staining (Fig.16B, E). Keratin 6 is expressed in activated keratinocytes of the hyperproliferative epithelium and in hair follicles (Fuchs, 1990; Wankell et al., 2001). Therefore, immunohistological analysis using keratin 6 antibodies was performed. These experiments demonstrated a reduction in the thickness of the hyperproliferative epithelium in the conditional Met mutant mice compared to controls (Fig.16C, F). Three days after injury, the hyperproliferative epithelium in the mutant mice consisted only of a few cell layers and was not dramatically different from the normal epidermis. However, in control mice, newly formed epithelium appeared much thicker. Five days after injury, the size of the hyperproliferative epithelium increased in both, control and mutant, but interestingly, in mutant it was more prominent than in the control (e.g. compare Fig.16B with 16E).

Figure 16. Wound healing in conditional Met mutant mice. A and D. Hematoxylin/eosin staining of sections of wound from control and mutant mice 3 days (A) and 5 days (C) after wounding. B and E Masson trichrome staining of sections 3 days (B) and 5 days (D) after injury. C and F. Immunofluorescence staining for Keratin 6 (red) and fibronectin (green) from control and mutant mice 3 days (E) and 5 days (F) after injury. Arrows indicate the hyperproliferative epithelium (HE). F, fatty tissue; G, granulation tissue; Es, eschar; HF, hair follicle Scale bar, 100μm

Thus, wound healing occurred in conditional Met mutant mice, but it was delayed and required about twice as much time as in control mice. The effect of wound closure was also determined as percentage of distance covered by the epidermis between the wound edges. For instance, 5 days after the injury, 50% wound closure was observed in control mice; in conditional Met mutant mice, 50% wound closure occurred in 9 days (Fig.17A). In control mice, most of the wounds were healed within 10 days, while in mutant mice it took 17 days. The formation of the hyperproliferative epithelium was indeed delayed during the repair process in the conditional Met mutant mice, as shown by quantification. Compared to control mice, the area of the hyperproliferative epithelium was reduced by 80% 3 days after injury; 5 days after injury the area was reduced by 65%, and 7 days after injury by 25% (Fig.17B). The dynamic of the growth of the wound epithelium however, was faster in the mutant than in the control, starting from day 3 after injury (Fig.17B). It was related to faster increase of cell numbers in the wound epithelium in the mutants, compared to controls (Fig.17E). In control wounds, amplification in cells number between day 3 and 5 after injury was 1.9 times, while in the mutant it was 3 times.

To test whether the delay in the formation of the hyperepithelium was correlated with keratinocyte proliferation, the numbers of 5-bromodeoxyuridine (BrdU)- and phospho-histone 3–positive keratinocytes in mutant and control wounds were counted. Indeed, the number of BrdUpositive nuclei was significantly lower in the mutant wound epithelium than in the controls (Fig.17F). However, the percentage of proliferating keratinocytes in the hyperproliferative epithelium 3 days after injury was increased in the conditional Met mutants, which could be related to the recovery of size of the hyperproliferative epithelium at later stages (Fig.17C, D). Proliferation-positive cells did not accumulate at any particular sites in the hyperproliferative epithelium or at the remnants of the hair follicles. Another possible explanation for the delayed formation of the hyperproliferative epithelium in conditional Met mutant mice was an increase in cell death. However, the number of apoptotic cells in the skin of control and mutant mice was comparable, as assessed by TUNEL staining. Thus, wound healing occurs in the skin of Met conditional mutant mice, but reepithelialization of wounds is delayed and requires about twice as much time as in control mice.

Figure 17. Quantification of wound healing in control and conditional Met mutant mice A. Wound closure kinetics in control and mutant mice. B. Quantification of the area of hyperproliferative epithelium 3, 5 and 7 days after wounding in control and mutant mice; only sections of the middle of the wounds were used for quantification. C. Proliferation of keratinocytes in the hyperproliferative epithelium from control and mutant mice 3, 5, and 7 days after wounding, as assessed by the proportion phosphohistone 3positive nuclei in the epithelium. Error bars represent standard deviations. A Student’s test was performed, and significant differences between control and mutant was observed 3 days after injury, P value, p=0.01. D. Proliferation of keratinocytes in the hyperproliferative epithelium from control and mutant mice 3, 5, and 7 days after wounding, as assessed by the proportion of BrdUpositive nuclei in the epithelium. Significant statistical differences between control and mutant was observed 5 days after injury, P value, p=0.01. E. Number of cells in the hyperproliferative epithelium quantified as Yopropositive cells for control and mutant 3, 5 and 7 days after injury. Yopro is a nuclear dye. F. Quantification of BrdUpositive cells in the hyperproliferative epithelium of control and mutant mice at different time points after injury.

Contribution of cells in the hyperproliferative epithelium

To assess if the conditional Met mutant cells (approximately 95% of the keratinocytes in the epidermis) were able to contribute to the newly formed epithelium of the wounds, hyperproliferative epithelia of many control and mutant wounds were collected by laser capture microdissection and analysed by Southern blotting. The laser capture microdissection allows for precise dissection of wound epithelium. An example of a section of a wound before and after microdissection is shown in Fig.18A, B. Importantly, Southern blot analysis revealed an absence of Metmutant cells, i.e. absence of the MetΔallele, in the microdissected hyperproliferative epithelia of mutant mice at day 5 (Fig.18C). Instead, all cells from the hyperproliferative epithelium of mutants contained the non-recombined Metflox allele, despite the fact that this cell population constituted only 5% in the skin prior to injury. At day 3, a 1:1 mixture of Metflox and MetΔ cells was seen, indicating that the wounded epithelium at early time points after injury consisted of recombined and the nonrecombined cells. In the unwounded mutant epidermis, only the recombined Metflox allele, i.e. MetΔ was detected.

Figure 18. Only residual Met positive keratinocytes contribute to the hyperproliferative epithelium of wounds in conditional Met mutant mice A, B. Isolation of hyperproliferative epithelium by laser capture microdissection. A wound section before (A) and after laser capture microdissection (B) is shown. C. Southern blot analyses of back epidermis and hyperproliferative epithelia from conditional Met mutant mice. Microdissected hyperproliferative epithelia of wounds 3 days (middle) and 5 days (right) after injury were collected. Southern blotting of two preparations from different pools of microdissected tissues is shown. The hyperproliferative epithelium 5 days after injury in conditional Met mutant mice is formed exclusively by cells, which contain the nonrecombined Metflox allele. At day 3, a 1:1 mixture of recombined and nonrecombined cells are seen (middle).

The hyperproliferative epithelium at day 5 was examined by immunofluorescence and immunohistochemistry using anti-phospho-Met antibodies. The data revealed that indeed, the majority of cells contained the activated Met receptor in both the control and mutant skin (compare Fig.19A, C with B, D). Positive Met staining was more pronounced in the upper, already differentiated layers of the hyperproliferative epithelium, but was also visible in lower layers of the epithelia (see arrows in Fig.19A-D). It should be pointed out that activated Met was almost undetectable in the normal epidermis away from wound in the mutant (left side of Fig.19B).

Figure 19. Only phosphoMet positive cells contribute to the hyperproliferative epithelium in control and mutant. Immunohistological analysis of wound sections from control and conditional mutant mice using anti-phosphoMet antibodies (red immunofluorescence in A and B, and brown immunohistochemistry in C and D). Cells in the hyperproliferative epithelium of conditional Met mutant mice (outlined) are phosphoMet positive. Arrows mark phosphoMet positive cells in the lower hyperproliferative epithelia layers. Scale bar, 100μm

It can be concluded from these data that only non-recombined keratinocytes, i.e. those that express a functional Met, can participate in the formation of the hyperproliferative epithelium. Thus, in the skin of conditional Met mutant mice the few remaining cells that escaped recombination appear to compensate and generate the entire hyperproliferative epithelia. Collectively, the data confirm that Met plays crucial functions during wound closure in the skin.

Scratch-wound healing of Met mutant keratinocytes in cell culture

The closure of scratchwounds in cultured primary skin keratinocytes in response to HGF/SF was analyzed to confirm the essential role of Met in wound closure also in cell culture. Primary keratinocytes were isolated from the skin of newborn control and conditional Met mutant mice (Caldelari et al., 2000). Immunohistological staining with keratin 14 antibodies indicated that the isolated cells from control and mutant mice corresponded to keratinocytes and were not fibroblasts or other cells (Fig.20A). To confirm the expression of Met in isolated keratinocytes, Nothern blot analysis was performed on control keratinocytes stimulated with HGF/SF using the Met probe. Indeed, isolated cells expressed the Met tyrosine receptor. This again demonstrated that keratinocytes, not fibroblast or other cells were cultured, since Met is expressed in epithelial cells.

Isolated primary keratinocytes from the skin of newborn control and conditional Met mutant animals were cultured, and monolayers were scratchwounded (Fig.21A-C, (Sano et al., 1999). In the presence of HGF/SF, control cells closed the wound within 48 hours. Keratinocytes isolated from Met conditional mutant mice did also close the scratchwounds in the presence of HGF/SF, but only within 96 hours. However, keratinocytes isolated from Met conditional mutant mice that were stimulated with TGFα, closed wounds already after 48 hours. A strong proliferative response towards HGF/SF was observed in control cells close to the wound edges at 24 hours, but such a response was not observed in the mutant keratinocytes (Fig.21D, quantification in 21E). 24 hours after scratching, keratinocytes isolated from mutant skin did not proliferate, only single cells were phosphohistone 3positive. The deficiency of proliferation in the culture of mutant keratinocytes stimulated with HGF/SF led to less density of cells close to the wounds, compared to control cultures (Fig.21D, compare control to mutant).

Figure 21 Scratchwound healing in cell culture of primary keratinocytes: Primary keratinocytes were isolated from newborn skin of control (A) and conditional Met mutant mice (BC). After scratchwounding, cells were further cultured in the presence of HGF/SF or TGFα. Photos were taken 0, 24, 48 and 96 hours after scratchwounding. Wounds in the cultures derived from conditional mutant mice did only close after 96 hours in the presence of HGF/SF. Scale bar, 100μm. D. Proliferation of primary keratinocytes from control and conditional Met mutant mice 24 hours after stimulation with HGF/SF, as assessed by phosphohistone 3 antibody staining (red). A dashed line marks the scratch edge. Counterstaining was performed with phalloidin (green). Scale bar, 100μm E. Quantification of proliferation of primary keratinocytes at wound edges stimulated with HGF/SF in the experiments described in D. Error bars represent standard deviations.

Next, the remaining non-recombined keratinocytes from the skin of conditional Met mutant mice were tested for their ability to contribute to wound closure in culture. Primary keratinocytes were stained with anti-phospho-Met antibody at different stages of scratch-wound closure. Phosphorylated Met could be detected at the membranes of control cells, and very rarely in keratinocytes isolated from conditional mutant mice 24 hours after cultured with HGF/SF (Fig.22A, B). When keratinocytes isolated from Met mutant skin were cultured for 48 hours and longer in the presence of HGF/SF, phosphoMet positive cells accumulated exclusively at the wound edges, and after 96 hours, the majority of the cells that had closed the scratchwounds contained phosphoMet (Fig.22B). Thus, as in skin wounds in vivo, the scratchwound area in the mutant culture were finally closed with Metpositive cells. Therefore, these data indicate that only Metpositive, nonrecombined cells, participate in wound closure in vitro and in vivo.

Figure 22 Only Met positive primary keratinocytes migrate into the scratchwounds in cell culture. A, B. Primary keratinocytes isolated from control (A) and conditional Met mutant (B) skin were scratchwounded and further cultured with HGF/SF. After 24, 48 and 96 hours cells were stained with phosphoMet antibodies (green). Nuclei were visualised by Yopro staining (red). In mutant cell population, phosphoMet containing cells were initially rare, but finally, after 96 hours, occupied the entire scratched area. The original edges of the scratchwounds are marked with a dashed line. Scale bar, 50μm

Cytoskeleton rearrangement in cultured scratchwounded keratinocytes

The properties of the cells at the wound edges were further examined by immunofluorescence staining for proteins that are important in directed cell migration like vinculin, paxillin, and VASP (Mitchison and Cramer, 1996; Rottner et al., 1999; Rottner et al., 2001; Raghavan et al., 2003). In the presence of HGF/SF, control keratinocytes showed increased numbers of focal adhesions as well as lamellipodia at the wound edges, and these structures pointed directly towards the wounds (Fig.23A, B, C, left pictures). Actin stress fibres, which were stained by phalloidin, were also oriented toward the wounds (Fig.23A, D). Control cells at the wound edges displayed a preferential location of RhoA staining at the rear of cells, and such localization is a characteristic feature of migrating cells (Fig.23D, left; see also Nobes and Hall, 1999; Raftopoulou and Hall, 2004). These control cells at the edges of the wound also reoriented their microtubules, which were demonstrated by γ-tubulin staining. The major arrangements of microtubules were not centrosomal in keratinocytes (Fig.23E). In contrast, Met mutant keratinocytes did not rearrange the proteins, which are known to be important during cell motility (Fig.23AE, right pictures). In the Met mutant cells, RhoA staining appeared punctuated cytoplasmatically, but was also perinuclear. Keratinocytes from conditional Met mutant mice displayed only few new focal contacts and stress fibers, and these were not oriented towards the wounds.

Figure 23. Met mutant keratinocytes are unable to rearrange their focal contacts and their cytoskeleton at the scratchwound edges following HGF/SF treatment Keratinocytes derived from control and conditional Met mutant mice were stained 24 hours after scratchwounding with antibodies directed against vinculin A., with phalloidin A, D, antibodies directed against VASP B, paxillin C, RhoA D and γtubulin E. Arrows mark the newly formed focal contacts (AC) and RhoA at the rear of the cells (D). Arrowheads mark cytoplasmatical and perinuclear localization of RhoA in mutant. The dotted line indicates the edges of the wounds. Scale bar, 50μm (AD), 20μm (E).

Signal transduction in primary keratinocytes

The isolated primary keratinocytes from the skin of control and conditional Met mutant mice were used to study signal transduction by molecules that are crucial for cell proliferation and cell migration (Rubin et al., 1991; Morimoto et al., 1991; Hartmann et al., 1994; Ridley, 2001; Khwaja et al., 1998). In the presence of HGF/SF and TGFα, such molecules like Erk1/2, Akt, Gab1 and PAK1/2 were activated in control primary keratinocytes (Fig.24A). In contrast, Erk1/2, Akt, Gab1 and PAK1/2 in mutant keratinocytes following stimulation by HGF/SF, were not activated. However, TGFα in mutant cells did activate these signaling molecules. The phosphorylation of PAK1/2 was quantified in control and mutant cells that were activated with HGF/SF or TGFα, and showed that stimulation with HGF/SF did not change the activation level of mutant keratinocytes, but did upon TGFα. The peak of PAK1/2 activation was observed after 10min stimulation of TGFα in control and mutant, and the same peak was detected after HGF/SF stimulation, but only for the control (Fig.24B).

Taken together, these in vitro data demonstrate that HGF/SF and Met signaling is important for the induction of proliferation and migration of primary keratinocytes in cell culture. Activation of this signaling pathway results in major reorganization of adhesion and cytoskeleton complexes like focal adhesions, lamellipodia, and stress fibers, which allows cells to move into the scratchwounds.

Figure 24 Signaling is blocked in keratinocytes derived from conditional Met mutant mice that are treated with HGF/SF, but not with TGFα. A. Western blot analysis of phospho Erk1/2, total Erk1/2, phospho Akt, total Akt, phospho Gab1 and phospho PAK1/2 in keratinocytes derived from control and conditional Met mutant mice. Cells were stimulated with HGF/SF or TGFαfor 0, 10 or 30 minutes. Erk1/2, Akt, Gab1 and PAK1/2 are not activated (phosphorylated) in cultured keratinocytes from the conditional mutant mice after HGF/SF stimulation. B. Quantification of the phosphoPAK1/2 signal on Western blots (A) as assessed by pixel intensity.